The nitrate ion is an oxidative an ion with the Molecular formula NO3− and a Molecular mass of 62.0049 g/mol. Nitrate represents the most oxidized chemical form of nitrogen found in natural systems. All living systems require nitrogen to exist since nitrogen is used to build many essential components such as proteins, DNA, RNA and vitamins, as well as hormones and enzymes. Higher organisms such as animals cannot use simple forms of nitrogen, such as nitrate and ammonium, and are instead dependent on complex forms of nitrogen such as amino acids and nucleic acids.
While nitrogen is an essential building block for life, nitrogen in the form of nitrate can also be harmful. When nitrate is taken in by eating food and drinking water, nitrate is converted to nitrite. Nitrite then combines with hemoglobin to form methemoglobin. This process can lead to the Hypoxia (medical) in organ tissue and a dangerous condition called Methemoglobinemia. Methemoglobinemia in infants is known as blue baby syndrome. Infants are more susceptible to nitrate toxicity than older children or adults. While fatalities are rare, sub-acute methemoglobinemia can affect development. Chronic consumption of high levels of nitrate may also cause other health problems, such as cancer or may contribute to disturbing the growth and development of an embryo or fetus. This is because excess nitrite moves into the bloodstream where it binds strongly to blood hemoglobin and impairs the delivery of oxygen to the embryo or fetus. Elevated levels of nitrate also lead to a build-up of nitrite in the gastrointestinal tract by nitrate reducing bacteria. There have recently been reports of a link between nitrate levels in drinking water and bladder cancer in women. Blood and serum nitrate levels can also become elevated as the result of increased production of nitric oxide (NO). Nitric oxide is an unstable gaseous compound that readily diffuses into body fluids where it can be converted to nitrate, nitrite or S-nitrothiol. NO levels rise during heightened immune-response such as occurs during sepsis, organ failure or graft-rejection.
Some adults can be more susceptible to the effects of nitrate than others. The Cytochrome b5 reductase Enzyme may be under-produced or absent in certain people that have an inherited mutation. Such individuals are unable to break down methemoglobin as rapidly as those that have the enzyme, leading to increased circulating levels of methemoglobin with the result that their blood is not as oxygen-rich. Those with insufficient stomach acid may also be at risk. Such individuals may include, for example, vegetarians and vegans. The increased consumption of green, leafy vegetables that typically accompanies vegetarian and vegan diets may lead to increased nitrate intake. While nitrate exposure is most easily caused by drinking water, it can also be caused by eating vegetables with high levels of nitrate. The high levels of nitrate in plants may be caused, for example, by growth conditions such as reduced sunlight, the undersupply of the essential micronutrients molybdenum (Mo) and iron (Fe), or high concentrations of nitrate due to reduced assimilation of nitrate in the plant. High levels of nitrate fertilization also contribute to elevated levels of nitrate in the harvested plant. A wide variety of medical conditions, such as food allergies, asthma, hepatitis, and gallstones may be linked with low stomach acid; these individuals may also be highly sensitive to the effects of nitrate.
Nitrate does not, however, only affect humans. Other animals are also affected. Nitrate can reach such high levels in some Freshwater or Estuary systems close to land so as to potentially cause the death of fish. Nitrate levels over 30 ppm can inhibit growth, impair the immune system and cause stress in some aquatic species. Supplying a nitrogen-limited eco-system with high levels of nitrate can result in significant increases in the levels of phytoplankton (algae) and macrophytes (aquatic plants). This can pose a significant threat to fragile ecosystems. The recommended level of nitrates to avoid the propagation of algal blooms is between 0.1 to 1 mg/L.
Nitrate is a wide spread contaminant of ground and surface waters worldwide. The accumulation of nitrate in the environment is greatly impacted by runoff from the over-application of nitrogen fertilizers. Nitrate contamination can also occur from concentrated animal feeding operations and from poorly or untreated human sewage. Because nitrate is a naturally-occurring chemical that is left after the breakdown or decomposition of animal or human waste, water quality may also be affected if a high number of septic systems exist in a watershed. Septics leach down into ground water resources or aquifers and supply nearby bodies of water. Lakes that rely on ground water are often affected by nitrification through this process. Nitrate-containing wastes are also produced by many industrial processes including paper and munitions manufacturing. The burning of fossil fuels in power plants and cars, SUVs and all internal combustion engines results in the production of nitric acid and ammonia as air pollution.
Nitrate ion from fertilizers, sewage and manufacturing has reached high concentrations in water supplies throughout the world. The analytical control of nitrate concentrations in surface waters, especially those which serve as drinking water sources, is therefore regulated in most advanced countries. The United States Environmental Protection Agency (EPA) has, for example, established an enforceable regulation for nitrate, called a maximum contaminant level (MCL), at 10 mg/L or 10 ppm. The EPA thereby notes that infants below six months who drink water containing nitrate in excess of the maximum contaminant level could become seriously ill and, if untreated, may die.
Because the major environmental release of nitrate arises from its use in fertilizers, it is unlikely that the nitrate problem will disappear anytime soon. A continued need to monitor nitrates in finished drinking water, watersheds, industrial wastewater, private wells and estuaries exists. Nitrate contamination of source water will also continue to be relevant for industries that depend on water purity for manufacturing their products.
In addition to other well-known specific nitrate determination methods, such as ion chromatography or direct potentiometry (so-called NO3 ISE or ion selective electrode), colorimetric nitrate determination methods currently serve as the “backbone” of nitrate analyses in water laboratories.
One of the current state of the art colorimetric methods is the LCK 339 nitrate kit provided by Hach Lange GmbH. The LCK 339 provides a highly accurate and reliable nitrate analysis for waste water, drinking water, raw water, surface water, soils, substrates and nutrient solutions in the range of 0.23-13.50 mg/L NO3—N with a cuvette path length of 11 mm. The LCK 339 determines the concentration of nitrate based on the principal that nitrate ions in solutions containing high concentrated sulfuric and phosphoric acids react with 2,6-dimethylphenol to form 4-nitro-2,6-dimethylphenol, which can in turn be detected colorimetrically at a wavelength of 340 nm.
The LCK 339 itself improves on known state of the art analytical methods whereby the concentration of NO3—N is colorimetrically determined using 2,6-dimethylphenol. However, said known methods only have a detection range of 0.5-25.0 mg/L NO3—N with a cuvette a path length of 10 mm at a wavelength of 338 nm and are thus inferior to the LCK 339 offered by Hach Lange GmbH.
The colorimetric detection of nitrate levels using the aforementioned methods has several disadvantages. For example, analytical methods using 2,6-dimethylphenol are sensitive to side reactions in the presence of chlorides. This leads to low NO3—N recoveries and places a cap on the detection limit. Samples containing chloride, calcium or nitrite salt can furthermore either not be analyzed or can only be analyzed in a limited fashion. Sea water, brackish water and/or water from municipal wastewater treatment plants having a high salt content therefore usually cannot be analyzed with the aforementioned methods. Nitrate in ultra-pure water and in drinking water having a nitrate concentration below the aforementioned detection limits can also either not be detected or detected only imprecisely using the aforementioned methods. Wavelengths of between about 340 nm and 370 nm are also susceptible to interference which negatively impact detection accuracy.
Other colorimetric nitrate methods such as the Spectroquant® cuvette test 114556 offered by Merck make use of a side reaction, the so-called Liebermann Nitroso Reaction (LNR), in the presence of chlorides to form an intense colored product. This reaction is mainly used for the colorimetric determination of phenols. The application of the LNR as a nitrate analytical method has the disadvantage that long reaction times of at least 30 minutes are necessary for the complete reduction of the nitrate to the nitroso compound if chloride is the sole reducing agent. Measuring earlier would yield imprecise and inaccurate results. Nitrate tests using the LNR generally have a measuring range limit of 0.1 to 3.0 mg NO3—N mg/L using a 16 mm cuvette. The variation coefficient of this method is, however, double that of the variation coefficient of the aforementioned nitrate analysis methods using 2,6-dimethylphenol. The 16 mm thickness of the cuvette is also almost 1.5 times thicker than the 11 mm cuvette used in the 2,6-dimethylphenol analysis method. The preciseness of this analysis method will therefore necessarily be less than that of the 2,6-dimethylphenol analysis method.
Other analytical methods exist to determine nitrate concentrations, however, all have certain disadvantages.
Nitrate concentration can, for example, be indirectly determined by reducing nitrate to nitrite. The reducing agent for this method is usually either granulated and/or activated cadmium or hydrazine salts, both of which are, however, highly poisonous, such that their sale and disposal is prohibited or limited in many countries for environmental reasons. Following reduction, the nitrite ions are then reacted with an aromatic amine to form a diazonium salt in the presence of an acidic buffer. The diazonium salt in turn reacts with N-(1-naphthyl)-ethylene-diamine to form a red-violet azo dye which allows a semi-quantitative visual comparison with a color scale to occur. Test strips, color cubes and color disks based on this principle are available. While easy to use, test strips and color cubes only allow nitrate concentrations to be determined at certain, limited, concentrations, such as, for example, at 0, 1, 2, 5, 10, 20 and 50 ppm NO3—N for the AquaChek® test strips from Hach Lang GmbH. The easy to use color disks allow for a slightly more accurate NO3—N concentration to be determined, but are also limited to an accuracy in the mg/L NO3—N range. The accuracy of the color disk NI-11 from Hach Lange GmbH, for example, is 0-50 mg/L NO3—N. A somewhat more precise evaluation of the absorption of the color test solution is also possible photometrically.
Nitrate concentration can also be accurately measured through complex high-end systems such as the NITRATAX® family of probes or the GANIMEDE laboratory analysers offered by Hach Lange GmbH. The NITRATAX® probes are specifically designed to constantly monitor the concentration of nitrate by direct immersion in activated sludge, wastewater and/or surface water. The NITRATAX® plus sc model, for example, has a very good measuring range of 0.1-100 mg/L NO3—N. The automated GANIMEDE N analyzer offered by Hach Lange GmbH can analyze for total nitrate concentration. The GANIMEDE N operates using reagents to digest unwanted by-products and to thereby provide a “pure” water sample for analysis. Direct UV measurement at approximately 210 nm versus a reference wavelength of approximately 228 nm, high-quality optics and a complex algorithm are used to calculate nitrate concentrations at a very good measuring range of 0.5-150 mg/L NO3—N (after digestion).